Current vaccines against Haemophilus influenzae type b have reduced the incidence of H. influenzae type b invasive diseases. However, they provide no protection against nontypable strains which remain a common cause of otitis media, the most common infection of childhood, and pneumonia. H. influenzae has an absolute growth requirement for heme and the human body is its sole niche. We have characterized several of the heme and iron acquisition systems of H. influenzae and shown that their expression is regulated by iron/heme levels in the growth media. In addition we have shown that the genes are expressed in vivo in both humans and animal models, indicating that the in vivo environment is similarly heme restricted. However our understanding of the systems involved in the acquisition of this essential nutrient and their regulation remains incomplete. We propose a genomics analysis to define the genes whose expression is regulated by iron and heme levels (the FeHm regulon) and to determine their role in virulence in animal models of disease. The FeHm regulon has been defined in one sequenced H. influenzae strain using microarray technology, and we will use the same approach to define the FeHm regulon in two additional sequenced strains. Following identification of the genes we will construct pools of signature-tagged mutants for analysis in vivo in three animal models (the chinchilla model of otitis media and nasopharyngeal colonization, the rat model of invasive disease and the rat model of persistence in the lung). Mutants will additionally be compared to wildtype strains for in vitro growth with various heme sources, as well as protection against heme toxicity and oxidative damage and for their ability to form biofilms. These studies will provide a framework for advancing our understanding of the iron/heme acquisition systems of H. influenzae with further potential for providing preventive and treatment strategies for H. influenzae disease.